Journal of Molecular Catalysis B-enzymatic最新文献

Isoquercitrin, a rare flavonol glycoside with wide biological activities and key synthetic intermediate for the production of enzymatically modified isoquercitrin (EMIQ), was conducted by naringinase-catalyzed conversion of rutin under ultrasound irradiation. The maximum yields were obtained to 98.35 ± 3.13% and 95.20 ± 2.52% under conventional heating and ultrasound irradiation, respectively. The optimal results under ultrasound irradiation were obtained under the following conditions: rutin concentration 0.8 g/L, naringinase concentration 3000 U/L, reaction temperature 40 °C for 20 min, which was more economical than that with conventional heating. The reaction time was reduced from 60 min to 20 min, and the apparent kinetic parameter (V_m/K_m) was increased 3.72-fold. The lower activity energy E_a under ultrasonic irradiation was 0.7-fold of that in an incubator reactor, which could easily initiate the enzymatic reaction. The association saturation constant K_a was 1.98-fold higher than that with conventional heating, showed a better affinity between rutin and naringinase detected by surface plasmon resonance (SPR) analysis. These results suggest that ultrasound irradiation can accelerate the enzymatic synthesis of isoquercitrin from rutin.

Phenylpropanoid glucosides (PPGs) are naturally occurring and bioactive phenolic derivatives, largely distributed in plants. In this work different PPGs have been chemically or enzymatically synthesized from the lignols coniferyl and p-coumaryl alcohols as substrates for a laccase-catalyzed oxidative coupling. The biooxidation of these PPGs has been investigated here and novel dihydrobenzofuran-based structurally modified analogues have been isolated and characterized. Specifically, the presence of a carbohydrate moiety increased the water solubility of these compounds and reduced the number of dimeric products, as pinoresinol-like structures could not be formed. Looking for a possible sugar-promoted stereochemical enrichment of the obtained diastereomeric mixtures of dimers, different carbohydrate moieties (d-glucose, l-glucose and the disaccharide rutinose) were considered and the respective d.e. values of the dimeric products were measured by ¹H NMR and HPLC. However, it was found that the sugar substituent had a minor effect on the stereochemical outcome of the radical coupling reactions, the best measured result being a d.e. value of 21%.

In this work, we explored the activation of a lipoprotein lipase from Burkholderia species by glucose-headed surfactants (GHSs) for enhancing its catalytic activity in organic solvent. Three GHSs were prepared and then tested as the additives for inducing the activation of lipoprotein lipase. The kinetic parameters of GHS-treated lipoprotein lipase were determined for the hydrolysis or alcoholysis of p-nitrophenyl acetate. It was found that GHS-treated lipoprotein lipase was 4 to 5 orders of magnitude more active than its native counterpart in organic solvent. Such a dramatic activity enhancement was largely the result of a huge increase in the turnover frequency k_cat. Surprisingly, the k_cat values in organic solvent were one order of magnitude greater than their aqueous counterparts. As a result, the k_cat/K_m of GHS-treated lipoprotein lipase in organic solvent became comparable to the aqueous level within one order of magnitude. We thus have demonstrated for the first time that a lipase can display nearly aqueous-like activity in organic solvent. As an illustrative application of GHS-treated lipoprotein lipase, we performed the dynamic kinetic resolution of two secondary alcohols, which provided the products of high enantiopurity (98–99%ee) with high yields (90–91%).

Lipase-based cross-linked aggregates were investigated for a non-specific reaction, i.e. the epoxidation of α-pinene to its oxygenated derivatives. The activity of the biocatalysts has been evaluated in a green context, i.e. ethyl acetate as both acetate-supplier and organic solvent with H₂O₂/UHP/TBHP as oxidant. Screening of the lipase sources indicated Aspergillus niger lipase as the most efficient biocatalyst for this reaction. Different immobilization protocols ((i) cross-linked enzyme aggregates (CLEA), (ii) cross-linked enzyme aggregates onto magnetic particles (CLEMPA) and (iii) covalent immobilized enzyme (CIE) onto magnetic particles (MP)) were evaluated considering the activity as main parameter. Thus, CLEA and CLEMPA afforded better epoxidation yields of α-pinene towards CIE. The investigated biocatalytic systems allowed to transform α-pinene into oxigenated derivatives with industrial and commercial applications (e.g. α-pinene oxide, camphene, pinanediol and camphonelic aldehyde). FTIR investigations on the biocatalysts revealed the effects of the immobilization protocol on the enzyme secondary-structure. Additionally, textural characterizations were performed by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) analysis.

The thermal stabilization of enzymes is a critical factor in the development and reliability of enzyme-based processes and functional materials. Using a simple amine coupling approach for enzyme immobilization onto magnetic microbeads, followed by encasement of the beads in a hydrogel, we demonstrate that the thermal stability of the enzyme acetylcholinesterase can be increased dramatically. For example, when free and microbead-immobilized enzyme (“EM Conjugate”) are incubated overnight in a dry state at 63 °C (140 °F), the catalytic efficiency (k_cat/K_m) of the latter is higher than the former by six orders of magnitude (a factor of 2.16 × 10⁶). This effect arises mostly through a ∼29,700-fold decrease in K_m experienced by the EM Conjugate, relative to that of the free enzyme. Encapsulation of the EM Conjugate in a hydrogel based on poly(N-(3-aminopropyl methacrylamide)), which contains a primary amine, affords the enzyme additional stability when incubated overnight at 63 °C in an aqueous state. For example, its catalytic efficiency is four orders of magnitude higher than that of both the free enzyme (a factor of 4.34 × 10⁴) and that of the EM Conjugate alone (a factor of 1.78 × 10⁴) after all are incubated overnight at 63 °C. The presence of the hydrogel also caused the Michaelis constant to decrease by 1.38 × 10⁴ relative to that of the EM Conjugate, reaching a value of 2.18 × 10⁻³ M. Thus the hydrogel enables the AChE substrate binding site to retain a significant amount of its natural affinity for the substrate, after heating. This effect may occur via ion-pairing by the primary amines in the hydrogel polymer repeat unit, which are protonated and positively-charged at the assay pH. To the best of our knowledge, this simple method for enzyme thermal stabilization is novel and has not yet been investigated.

Laccase-catalyzed synthesis of dye molecules represents a green choice to reduce the environmental footprint of conventional synthetic processes. Textile industry will benefit from this green technology since the synthesized dyes can be exploited to colour different fabrics.

This work describes the application of the Pleurotus ostreatus POXA1b laccase in polymeric dye synthesis using resorcinol and 2,5-diaminobenzenesulfonic acid (2,5-DABSA) as substrates. The potential of the resorcinol/2,5-DABSA coupling route was transferred to a chemical industry, Setaş Colour Center, by introducing a greener synthesis step within the process routinely used for textile dyeing. Dye synthesis was performed at different precursor ratios (1:1 and 1:10 2,5-DABSA: resorcinol) and their dyeing properties were compared on different fibres. The two mixtures of synthesized dyes proved to be effective on nylon and wool, with 1:10 ratio displaying the best performances in terms of dyeing efficiency and colour strength. Good and comparable end quality and “performances during use” were observed for nylon and wool coloured with both synthesized dyes.

This paper describes the enzyme immobilization protocol as well as the enzymatic method for the direct resolution of (R,S)-atenolol. The used magnetic enzyme carriers possess on their surface new-synthetized chitosan derivatives with free amine groups distanced by ethyl or butyl chain. Additionally the catalytic activity of two types of commercially available lipases from Candida rugosa immobilized onto two different magnetic nanoparticles were compared. The highest values of enantioselectivity (E = 66.9), enantiomeric excess of product (ee_p = 94.1%) and conversion (c = 41.84%) were obtained by using lipase from Candida rugosa OF immobilized onto Fe₃O₄-CS-EtNH₂. The study confirmed that even after 5 reaction cycles the immobilized lipase maintain its high catalytic activity.

Cold-active lipases have emerged as an important class of biocatalysts for chemical and food industries due to their high efficiency at low temperature and long-chain substrate preference. In an effort to explore the feasibility of converting a cold-active esterase from Monascus ruber (Lip10) into a cold-active lipase, an Y264F variant in which the salt bridge between K243 and Y264 was disrupted has been constructed and characterized. The interfacial kinetic parameter, K_m^app for pNP-laurate (C12) and pNP-palmitate (C16), of Lip10 esterase was 4.2 and 5.7 times higher than those of the Y264F variant, respectively. Substrate specificity of the Y264F variant changed from shot-chain length substrate to medium- and long-chain length substrates, indicating that the Y264F variant turned into a lipase. Meanwhile, the Y264F variant displayed 48.6% maximum activity at 4 °C and 3.2 kcal/mol activation energy in the range of 5–30 °C, suggesting that it was still cold-active. Based on analysis of the structure-function relationships, it suggests that the shape of substrate channel controlled by the conserved salt bridge was very important for the substrate specificity. This study provides a way to alter the substrate preference of the Lip10 esterase as well as new insight into the structural basis of esterase substrate specificity.